1. Technical Field
This application concerns a substituted, non-coding nucleic acid molecule for modulation of the activity of the human or animal immune system, as well as a method to make such molecule and a vaccine containing the substituted, non-coding nucleic acid molecule.
2. Background Information
In the formation of an immune response against an antigen, two major branches of the immune system are distinguished: on one side, the humoral arm, which relies on the synthesis of antibodies by B lymphocytes, but also a humoral component of non-adaptive immunity, the complement system. On the other side, the cellular arm relies on the activity of T lymphocytes, NK cells and antigen presenting cells of the immune system. T lymphocytes are capable of recognizing cells that are infected by virus. Correspondingly, the cellular arm is also referred to as the TH1 response and the humoral arm as the TH2 response.
Bacteria and parasites that predominantly exist extracellularly are usually fought by the TH2 arm. Pathogens that predominantly reside intracellularly, as is the case for some bacterial species and all viruses, however, are mainly fought by the TH1 arm, mainly meaning by cytotoxic cells.
The development of vaccines has continuously delivered vaccines that are more efficacious and at the same time show lesser side effects. Departing from attenuated live vaccines or inactivated pathogens, vaccines were developed that consist of antigens made by recombinant methods (for example against Hepatitis B virus, recombinant vaccines against herpes simplex virus, human papilloma virus (cervical carcinoma) and borrelia are in clinical development). These vaccines lead to a predominantly TH2 dominated immune response and need the co-application of adjuvants in order to attain stable immunity. These adjuvants thus lead to a balance of humoral and cellular immune response.
Some infectious diseases can only be prevented by vaccination, if the vaccine elicits a TH1 dominated immune response. For such vaccines, the combination of a DNA vaccine (induction of cellular immunity) with a recombinant antigen (induction of humoral immunity) is currently regarded as the optimal combination (Esteban et al., Vaccine 2002, 20: 1226-1231). The recombinant protein, which is difficult and costly to synthesize, is the decisive cost factor in this process. Peptides, which are less expensive to make, are only of limited utility since they induce a shift towards a TH2 dominated immune response.
Despite all progress in DNA vaccination, a real break-through has not been achieved until today. Several reasons exist for this fact, among them insufficient transfection and a resulting low level of expression of the vaccinated antigen, as well as a globally insufficiently pronounced immune response as a result of the vaccination.
In order to improve the transfection efficiency, different peptides and other organic molecules have been linked to gene transfer vehicles by different methods of coupling. Also, it was tried to utilize ligand-receptor interactions for improved uptake of gene transfer vehicles by coupling of ligands (Fraser et al., 1998, Semin. Immunl., 10 (5): 363-72).
By attaching the nuclear localization signal (NLS) of the SV-40 virus covalently to expression cassettes encoding the small surface antigen of Hepatitis (HBsAg), a 10 to 15-fold increased antibody titre could be demonstrated after intramuscular application (Schirmbeck et al., J. Mol Med. June 2001; 79 (5-6):343-50).
In order to avoid the disadvantages of current viral and plasmid-based gene transfer systems, covalently closed, minimalistic DNA expression constructs were developed (see EP 0 914 318 B1). These minimalistic expression constructs can also be linked covalently to a nuclear localization signal of the SV-40 virus and then lead to a shift towards a TH1 accentuated immune response (see WO 03/0031469).
In order to generate an improved immune response, CpG oligonucleotides (CpG-ODN) were employed as a new class of immune modulating molecules. Such unmethylated CpG motifs occur in bacterial DNA and constitute a danger signal for the immune system. As a pathogen associated molecular pattern (PAMP), they mainly result in an unspecific activation of the innate immune system (Krieg, Nat. Med 2003, 9: 831-835).
CpG ODN also induce a TH1-biased immune response by means of the cytokines interleukin-12, interferon gamma and tumor necrosis factor alpha.
Immune stimulatory nucleic acid sequences (ISS) that comprise the described CpG ODN are only a few bases in length and do not function through the expression of proteins encoded thereon.
The ISS used herein are dumbbell-shaped, covalently closed deoxyribonucleic acid molecules. They consist of oligonucleotides, the bases of which can partially form pairs with each other, and one or two hairpin loops that comprise 30 bases and contain several CpG motifs (see EP 1 196 178). It was demonstrated that the immune modulating effect of these dumbbell-shaped covalently closed deoxyribonucleic acid molecules is strongly dependent on the loop-double strand combination and the CpG motifs contained therein. Linearization, deletion of a hairpin loop, downsizing of the molecule or the removal of CpG motifs lead to a changed pattern of induction of cytokines.
The strong stimulation of the cellular immune response facilitates an influence on regulatory cycles that would, in the absence of interference, not lead to an immune activity that would be satisfactory to the patient.
These dumbbell-shaped, covalently closed and non-coding nucleic acid molecules, which comprise at least one or more CpG motifs and single stranded hairpin loops on one or both ends, are referred to synonymously as “carrier molecules” in the following.
In this context, the term “nucleic acids” is to be understood as meaning deoxyribonucleic acids.
Immune stimulation in the context of this invention means that the mediator and effector cells of the immune system, i.e. in particular the currently known thymocytes that possess a helper function, and cytotoxic thymocytes, B cells and so-called NK (natural killer) cells, macrophages and monocytes, as well as dendritic cells and their precursors, as well as functionally as yet unexplained cell populations with functions within the immune system, as described in the invention, are stimulated by nucleic acid molecules in order to proliferate, migrate, differentiate or become active. Immune modulation means that, besides providing general stimulation in the above-defined sense, the nature or character of an immune reaction is influenced, either by affecting an immune reaction that is currently still developing or maturing, or by modulating the character of a reaction that has already established itself.
Short nucleic acid molecules in the context of the present invention are such molecules having a chain length as given in the examples below (preferably 48 to 116 nucleotides).